A commercial matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) instrument equipped with a curved field reflectron (CFR) was modified in order to perform collision-induced dissociation (CID) on a variety of biomolecules. The incorporation of a high-resolution ion gate together with a collision cell within the field-free region allowed tandem mass analysis (MS/MS), without the necessity to decelerate the precursor ions prior to activation. The simultaneous detection of all product ions remained possible by using the CFR. To test the MS/MS performances, ACTH (fragment 1-17), a complex high mannose carbohydrate (Man)(8)(GlcNac)(2) and a lysophosphatidylcholine lipid (18:1) were analysed on the modified instrument. Direct comparison with the low-energy product ion spectra, acquired on a MALDI quadrupole ion trap (QIT) two-stage reflectron time-of flight (ReToF) mass spectrometer, showed significant differences in the types of product ions observed. The additional ions detected were a clear indication of the high-energy fragmentation processes occurring in the collision cell.
Matrix-Assisted Laser Desorption:lonisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) as a tool for differentiating bacterial species was examined using reference strains representing gram-positive and gram-negative taxa. Initially, the effect of differences in medium composition on spectral pro le was examined. The results indicated that growth on Columbia blood agar resulted in a larger spectrum of ionized residues and was therefore used for the cultivation of all strains in the rest of the study. The stability of the obtained mass spectral pro les against differences in batch and media processing suggested that no signi cant alterations to the pro les occurred in response to changes in media sources. The established conditions from these initial experiments were used to standardize subsequent experiments. The MALDI-TOF-MS pro les of 15 reference strains were compared and species characteristic markers were identi ed. The potential of using MALDI-TOF-MS as a tool for probing clonal diversity was examined using well characterized but clonally variable isolates of Bacteroides fragilis. Comparative analysis of the pro les of 20 strains revealed 5 clusters within the species but compared to other taxa such as Bacteroides merdae and Salmonella arizonae they form a closely related lineage. These results obtained strongly support the potential to use MALDI-TOF-MS as a tool for exploring bacterial surfaces for characteristic biomarkers and species-speci c signatures.
Many biological phenomena, such as cell cycle progression are controlled by networks of interacting protein complexes rather than by individual proteins. Advances in mass spectrometric identification of proteins, genomic sequencing and gene manipulation technology now allow to suggest a generic strategy to unravel protein interaction networks. In model organisms like S.cerevisiae genes can be rapidly tagged by a sequence encoding for an epitope recognized by monoclonal antibodies. Protein complex comprising the tagged protein can be isolated by a single step immunoprecipitation (IP). Subunits of the complex are separated by gel electrophoresis and identified by mass spectrometry. At first iteration of the strategy core subunits of the investigated complex are tagged, IP and yet unknown associated proteins are identified. At second stage newly identified proteins are also tagged, IP, and next generation of identified associated proteins is adjoined to the network. If biochemical data suggest that other proteins might be implicated in the process, such proteins can be tagged and linked to the network if found to be co-IP with any of core subunits of the complex. We applied this strategy to dissect the yeast Anaphase Promoting Complex (APC), a multi-subunit of ubiquitin-protein ligase implicated in cell cycle control in all eukaryotes. By using the strategy five new subunits of the APC were identified. Additionally, an evidence of physical interaction between APC and Cdc2Op was provided thus linking APC and spindle checkpoint protein complex.Matrix assisted laser desorption ionisation mass spectrometry (MALDI MS) is an established method for protein identification based on the approach of peptide mass fingerprinting. At the simplest level, the products of proteolytic digestion of a target protein are analyscd in linear time of flight (TOR mode without prior purification or sample dean up. As only singly charged molecules are observed in M U 1 MS. very complex mixtures can be analysed. The resulting mass signals are dependant on the sequence of the protein and therefore represent a unique pattern characteristic of the original target protein. The peptide maSSeS can then be applied to any one of a number of Web-based scarch engines that use protein sequence databases in order to identify the protein I. with the increasing number of protein sequences that are being entered into the databases. as well as putative translations from DNA fragments. the ability for unambiguous identification has also decreased. Clarification can be obtained by including post source decay (PSD) fragment data. generated in reflectron TOF. from one or more parent ions detected in the peptide spectrum. These additional masses then allow unequivocal protein identification'.Generating PSD data, however, is generally viewed to be time-and sampleconsuming. as fragment ions of different molecular weight need to be refocused to the deteetor to give a complete spectrum. A MALDI system. which employs both linear TOF and the unique curved field reflectr...
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